Mutual induced-fit mechanism drives binding between intrinsically disordered Bim and cryptic binding site of Bcl-xL

The intrinsically disordered region (IDR) of Bim binds to the flexible cryptic site of Bcl-xL, a pro-survival protein involved in cancer progression that plays an important role in initiating apoptosis. However, their binding mechanism has not yet been elucidated. We have applied our dynamic docking protocol, which correctly reproduced both the IDR properties of Bim and the native bound configuration, as well as suggesting other stable/meta-stable binding configurations and revealed the binding pathway. Although the cryptic site of Bcl-xL is predominantly in a closed conformation, initial binding of Bim in an encounter configuration leads to mutual induced-fit binding, where both molecules adapt to each other; Bcl-xL transitions to an open state as Bim folds from a disordered to an α-helical conformation while the two molecules bind each other. Finally, our data provides new avenues to develop novel drugs by targeting newly discovered stable conformations of Bcl-xL.


Note S1
Multicanonical MD algorithm S3 Fig. S1 Overview of role of Bim and Bcl-xL in the apoptosis process S4 Fig. S2 Multicanonical potential energy distributions of Bim in isolation using two forcefields S5 Fig. S3 Free energy landscapes of Bim in isolation using two forcefields S6 Fig. S4 Multicanonical potential energy distribution of Bcl-xL -Bim dynamic docking S7 Fig. S5 3D structure of picked representative configurations and the experimental structure S8 Fig. S6 Crystal packing observed in the experimental structure S10 Fig. S7 3D structure of picked representative configurations and S11 Fig. S8 Overview of binding pathway obtained from the multicanonical ensemble S13 Fig. S9 Location of residues that form the distance pairs used for pocket size analysis S14 Fig. S10 Reweighted distributions of distances of five pairs of residues S15 Fig. S11 Comparison between Bcl-xL -Bim complex structures from r1, r6 and r8 S16 Fig. S12 Comparison between Bim helix propensity between Bim in isolation and in the presence of Bcl-xL S17

Fig. S13
Free energy landscapes of Bim in isolation using AMBER ff99SB-ILDN with OPC waters S18

Fig. S14
Comparison of chemical structures. S19 Table S1 Results from Bim sampling simulations (in isolation) with the AMBER ff14SB force field with TIP3P waters S20

Table S2
Results from Bim sampling simulations (in isolation) with the AMBER ff99SB-ILDN force field with TIP3P waters S22

Table S3
Convergence of McMD dynamic docking simulations S25 Table S4 McMD-based dynamic docking results using subsets of the simulation data S27 Table S5 Per-residue R-values of Bim during 400 K canonical simulations S29 Table S6 Picking statistics from the McMD ensemble to produce the binding pathway for the path sampling simulations S30

Table S7
Distance between Cα atoms of residue pairs in structures rk and S31 Table S8 Results from Bim sampling simulations (in isolation) with the AMBER ff99SB-ILDN force field and the OPC water force field S32

Table S9
System & simulation parameters for Bcl-xL -Bim binding simulations. S36 Supplementary References S37 S3 Note S1: Multicanonical MD algorithm We used our own developed McMD-based dynamic docking method that has been thoroughly described in various previous papers, [1][2][3][4][5][6][7][8][9] but here we will shortly review the algorithm. The probability distribution of the potential energy of the multicanonical ensemble is defined by the following equation: where E is the potential energy, T0 the simulation temperature, n(E) the density of states and Zmc the partition function: W(E) is a weighting function to modulate the probability distribution Pmc in order for it to become constant and enables the system to take a random walk along the target energy range, and is defined as follows: ( ) = ln ( ) = + ln ( , ) where R is the gas constant and Pc the canonical energy distribution at T0. During the McMD simulations, this weighting function is used to scale the forces by a factor of ∇W(E), where the multicanonical temperature Tmc, which corresponds to T0/∇W(E), is restricted to a specific target range between Tlow to Thigh, which we generally set at 280 K and 700 K, respectively. Multiple iterations of sampling are required to estimate the correct bias that enables a random walk along a wide energy range, where the weighting function is updated between iterations using: After obtaining a flat potential energy distribution, a production run is executed to sample phase space. Due to the bias applied during the McMD simulations, the resulting multicanonical ensemble must be reweighted to obtain the canonical distribution at room temperature. A multicanonical distribution can be reweighted to a canonical distribution at any given temperature T within the flat energy range using the following equation: S5 Fig. S2. Multicanonical potential energy distributions of Bim in isolation using two forcefields. Potential energy probability distribution (PMcMD(E)) as sampled during the production run. Also shown are the reweighing canonical distributions (Pc(E, T)) at 300 K, 500 K and 700 K in blue, yellow and red, respectively. a) Distribution obtained using the AMBER ff14SB forcefield. b) Distribution obtained using the AMBER ff99SB-ILDN forcefield.

Fig. S3
. Free energy landscapes of Bim in isolation using two forcefields. a) Free energy landscape obtained using the AMBER ff14SB forcefield. b) Free energy landscape obtained using the AMBER ff99SB-ILDN forcefield. In both cases, the X indicates the location of the experimental structure (Bim taken from PDB ID 4QVF). The PCAs were performed independently from each other.
S7 Fig. S4. Multicanonical potential energy distribution of Bcl-xL -Bim dynamic docking. Potential energy probability distribution (PMcMD(E)) as sampled during the production run. Also shown are the reweighing canonical distributions (Pc(E, T)) at 300 K, 500 K and 700 K in blue, yellow and red, respectively.    S9. Location of residues that form the distance pairs used for pocket size analysis. Shown is the holo complex structure between Bcl-xL (white) and Bim (blue-red gradient). Indicated are the Cα atoms of Leu112 -Ser122 (d0), Leu108 -Val126 (d1), Phe105 -Leu130 (d2), Tyr101 -Arg139 (d3) and Glu96 -Asn136 (d4) that form the distance pairs along the surface of the pocket where Bim binds. S15 Fig. S10. Reweighted distributions of distances of five pairs of residues. Shown are the reweighted (300 K, 500 K and 700 K) probability distributions of distances between the Cα atoms of Leu112 -Ser122 (d0), Leu108 -Val126 (d1), Phe105 -Leu130 (d2), Tyr101 -Arg139 (d3) and Glu96 -Asn136 (d4). These pairs are along the surface of the binding site where Bim binds. The values corresponding to each of the structures rk are shown in Table S5.    1.000 0.00 a Characteristics for representative structure of obtained from the Bim conformational sampling using the AMBER ff14SB forcefield with TIP3P waters. Shown are, the relative cluster free energy (CFE) value in kcal/mol of the corresponding cluster k, the fraction of the ensemble population corresponding to the cluster in percentage, the first two principal components (PC1, PC2) in Fig. S2A, the fraction of the relative accessible surface area (RASA) of the peptide, the R(native)-value and the RMSD in Å of the heavy peptide atoms with respect to the experimental structure. PCA was performed on the distance matrix between the Cα atoms of Bim, excluding i±2 residues using data from both force fields. Subsequently, K-means clustering (k=1000) was performed on the PC coordinates (PC1-PC32) for this force field, followed by R-value scoring of the representative structures from the K-means clusters, where finally the clusters and their representative structures with a cluster free energy (CFE) of 2.5 kcal/mol were retained. a Characteristics for representative structure of obtained from the Bim conformational sampling using the AMBER ff99SB-ILDN forcefield with TIP3P waters. Shown are, the relative cluster free energy (CFE) value in kcal/mol of the corresponding cluster k, the fraction of the ensemble population corresponding to the cluster in percentage, the first two principal components (PC1, PC2) in Fig. S2B, the fraction of the relative accessible surface area (RASA) of the peptide, the R(native)-value and the RMSD in Å of the heavy peptide atoms with respect to the experimental structure. PCA was performed on the distance matrix between the Cα atoms of Bim, excluding i±2 residues using data from both force fields. Subsequently, K-means clustering (k=1000) was performed on the PC coordinates (PC1-PC31) for this force field, followed by R-value scoring of the representative structures from the K-means clusters, where finally the clusters and their representative structures with a cluster free energy (CFE) of 2.5 kcal/mol were retained.       The algorithm starts at window where λ = 0 Å with the structure , and then moves to window +1, picking one representative structure for this window that is similar to . Then, the process is repeated for the window +2, picking a structure similar to that of the preceding window, i.e. those from +1. Shown are the similarities between the picked structure from the preceding window to the current window, in terms of the R-value and the RMSD of the ligand. Finally, the cutoff used is listed, where "R:" corresponds to the R-value cutoff and "X:" to the RMSD based cutoff, where matching structures only have to fulfill one of the criteria and the number of matching structures is listed in the final column "N". In case the number of contacts is less than 25, the R-value cutoff is no longer used (in these cases, R:0.0 is set)